Osteopontin is an acidic, highly phosphorylated, sialic acid rich, calcium binding protein. Osteopontin contains approx. 28 moles of bound phosphate per mol osteopontin and binds approx. 50 moles of Ca per mole osteopontin.
Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes, such as bone remodelling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. Supporting an inhibitory function of OPN in ectopic calcification, an in vivo model using OPN-deficient mice showed diminished calcification upon exogenous addition of the protein. In addition, OPN is involved in the urinary tract's defence against the formation of renal stones because OPN can inhibit growth and aggregation of calcium oxalate monohydrate crystals.
The biological role of OPN in milk is not clear; however, several functions could be hypothesized. Osteopontin has been reported to be involved in mammary gland development and differentiation, and high levels of OPN expression have been observed in the mammary gland in early lactation. Furthermore, the highly anionic nature of the protein could enable OPN to form soluble complexes with calcium ions and thereby inhibit unintentional calcium crystallization and precipitation in milk.
In the scientific literature osteopontin is typically purified from bone or milk and it is typically present in bovine milk in a concentration of 20 mg/L. In milk, osteopontin is a serum protein but may also to some extent associate with the casein micelles depending on the Ca2+ level. Acid whey is the preferred raw material for industrial production of osteopontin. When acid whey is formed osteopontin is thought to leave the casein micelles as Ca2+ leaks out into the serum phase. This aspect makes acid whey a straightforward source of osteopontin. For the same reason sweet whey has a slightly lower osteopontin content. Furthermore, sweet whey contains caseino macropeptide (CMP) from enzymatic cleavage of the kappa-casein. CMP has many biochemical resemblances with osteopontin—both are small, flexible, acidic, phosphorylated glycoproteins. For this reason CMP and osteopontin is believed to be quite similar in their binding to ion exchange resins, which will pose a problem in purifying osteopontin from a CMP-containing raw material. Another aspect is the likely degradation of osteopontin by proteolytic enzymes used for cheese making. These three aspects may have resulted in avoidance of this raw material for osteopontin purification, both for industrial production and for scientific research.